First three new species of free-living marine nematodes of the Molgolaimus (Nematoda: Desmodoridae) from the continental shelf of the Brazilian coast (Atlantic Ocean)

Material and methods

Study area and sampling. This information were previously described in Manoel, Neres & Esteves (2024). Table 1 presents details of the collection stations relevant to this study. For sediment collection, a box-corer was used, while meiofauna samples were collected with a corer with dimensions of 10 cm × 10 cm. The sediment samples were taken in triplicate. The material collected was placed in plastic pots and fixed with 4% formaldehyde.

Sample processing. In laboratory, sediment samples were washed with filtered water through sieves of 0.5 mm and 0.045 mm mesh. The remaining samples in the smaller mesh sieve were extracted with SICOL-40 colloidal silica solution (specific gravity 1.18) (Somerfield, Warwick & Moens, 2005).

Under a stereomicroscope, Nematodes was sorted out and deposited in a small glass container containing Solution 1 described by De Grisse (1969). The specimens were then transferred to glycerin and diaphanized using the methodology described by De Grisse (1969), and subsequently mounted permanently on glass slides (Cobb, 1920). At the generic level, Nematodes were identified using keys provided by Warwick, Platt & Somerfield (1998) and Decraemer & Smol (2006). At a specific level, the identification was performed by comparing the characteristics of the species found with those mentioned in the original descriptions. Drawings and photographs were produced utilizing an Olympus CX 31 optical microscope equipped with a drawing tube. Body measurements were obtained with the assistance of a mechanical map meter.

For scanning electron microscopy (SEM), individuals were obtained by disassembling glycerin-paraffin slides. These specimens were rehydrated with distilled water according to the method described by Abolafia (2015). The individuals were then placed in a meiofauna processing manufactured container described by Abolafia (2015), gradually dehydrated in a graded ethanol series (10% during one day and 20, 30, 40, 50, 60, 70, 80, 90, 92, 95, first 100 and second 100% along the second day, changing from one concentration to the following every 2 h) and dried in a critical-point dryer. The specimens were removed from the container, deposited on an aluminum stub covered with conductive tape, coated with gold and examined under a TM4000 SEM at 10 kV with BSE detector.

The holotype and one female paratype for each species are deposited in the Nematoda Collection at the Museum of Oceanography Prof. Petronio Alves Coelho (MOUFPE) in Brazil. Additional paratypes are deposited in the Meiofauna Laboratory within the Zoology Department at the Federal University of Pernambuco (NM LMZOO-UFPE).

The electronic edition of this article, formatted in Portable Document Format (PDF), constitutes a published study in accordance with the guidelines set forth by the International Commission on Zoological Nomenclature (ICZN). Consequently, the new names presented in this electronic version are considered effectively published under the Code based solely on the electronic edition. This research, along with its nomenclatural actions, has been duly registered in ZooBank, the ICZN’s online registration system. The ZooBank LSIDs (Life Science Identifiers) can be accessed and the related information can be viewed using any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:01681568-8A01-47FC-96E5-DE43C7529F14. The online version of this research is preserved and can be accessed through the following digital repositories: PeerJ, PubMed Central, and CLOCKSS.

Systematics

Diagnosis. (After Leduc, Fu & Zhao, 2019) Cuticle finely striated, but may appear smooth under light microscopy (prominently annulated in M. pecticauda). Inner and outer labial papillae are small and often challenging to differentiate. Cephalic setae positioned slightly anterior or posterior to the head constriction. Amphidial fovea round and situated posterior to the head constriction. Buccal cavity small and weakly cuticularized, featuring small teeth that may sometimes be indistinct. Pharynx cylindrical, narrow and characterized by a prominent posterior bulb, typically spherical. Pharyngeal lumen weakly cuticularized, except within the pharyngeal bulb, where it may be heavily cuticularized. Secretory-excretory pore located anterior to the nerve ring and rarely found posterior to it. Female didelphic-amphidelphic, with reflexed ovaries (position of the genital branches varibles). Male monorchic with single anterior testis (variable position of the genital branch: on the right, on the left or positioned ventrally in relation to the intestine). Spicules of variable length and shape. Gubernaculum present or absent, when present with or without apophysis. Precloacal supplements often observed. Tail of varying shape and length (short and conical to elongated and conico-cylindrical).

Type species: Molgolaimus tenuispiculum Ditlevsen, 1921

Valid species (Table 2)

The valid species were divided into groups and, when relevant, into subgroups following the classification by Fonseca, Vanreusel & Decraemer (2006) with modifications in subgroups 1b₁, 1b₂, 4a and 4b (Table 2). The list of valid species is in accordance with Leduc, Fu & Zhao (2019), but with the addition of species described later and species described by Bussau (1993). The species described by Bussau (1993) are currently considered valid (Holovachov, 2020). Valid species for which synonyms occur are listed in Appendix S1.

Invalid species

The species listed below are considered taxon inquirendum for being poorly described (Jensen, 1978).

Description of new species

Type material. Four males and two females found. Holotype male (MOUFPE 0026), paratype female 1 (MOUFPE 0027), 3 male paratype (493–495 NM LMZOO-UFPE) and paratype female 2 (496–497 NM LMZOO-UFPE).

Type locality. South Atlantic Ocean, Continental shelf of the State of Bahia, Brazil, station 23 (S13°04′10.32″ W38°25′46.98″), December 11, 2019, 65 m.

Locality of paratypes. Paratype female 1: South Atlantic Ocean, Continental shelf of the State of Rio Grande do Norte, Brazil, station 2 (S05°42′54.42″  W34°59′31.92″), November 28, 2019, 60 m. Paratype males (1–3): South Atlantic Ocean, Continental shelf of the State of Sergipe, Brazil, station 17 (S11°00′00.54″  W36°49′58.98″), December 12, 2019, 54 m. Paratype female 2: South Atlantic Ocean, Continental shelf of the State of Sergipe, Brazil, station 16 (S10°44′59.28″  W36°25′32.88″), December 09, 2019, 58 m.

Etymology. The spicules of Molgolaimus sigmoides sp. nov. has a S-shaped structure. In Greek, sigma (ς) corresponds to the letter S in the Latin alphabet.

Holotype male. Body cylindrical 376.5 µm long. Maximum body diameter corresponding to three times the head diameter. Cuticle faintly striated. Somatic setae not observed. Head slightly set off from the rest of the body by a slight constriction. Inner and outer labial sensilla indistinct. Four short cephalic setae (<two µm long) located at the level of buccal cavity and slightly anterior to head constriction (three µm from anterior end). Amphidial fovea circular, located 10 µm from anterior end (1.7 times the head diameter) and occupying 53% of corresponding body diameter. Buccal cavity small, narrow, with lightly slightly cuticularized walls. Three small teeth, difficult to see (a slightly larger dorsal tooth and two smaller ventrosublateral). Pharynx muscular (73 µm long), surrounding buccal cavity, consisting of narrow, cylindrical anterior portion and with conspicuous spherical posterior bulb (81% of corresponding body diameter). Pharyngeal lumen slightly cuticularized, except in the pharyngeal bulb where the valves are more cuticularized. Nerve ring situated at 59% of the pharynx length from anterior end. Secretory-excretory pore not observed. Ventral gland located posterior to the pharyngo-intestinal junction. Cardia partially surrounded by intestine. Reproductive system with single anterior outstretched testis to the left of the intestine. Spicules S-shaped, arched ventrally at the anterior end and dorsally at the posterior end (2 times the cloacal body diameter). Gubernaculum surrounding the spicules at the distal end and with dorsal-caudal apophyses. Precloacal supplements absent. Caudal glands not observed. Tail conico-cylindrical, about four times the cloacal body diameter. The cylindrical portion of the tail represents about 18% of its total length. Spinneret short.

Paratype female. Similar to male. Body measuring 397 µm in length, with a maximum diameter of 15 µm (three times the head diameter). Amphidial fovea, occupying 39% of corresponding body width and located 10 µm from anterior end. Basal bulb occupying 85% of the corresponding body diameter. Nerve ring situated at 58% of the pharynx length, from anterior end. Vulva located 170 µm from anterior end, at 43% of body length. Reproductive system didelphic, with reflexed ovaries. Anterior gonad situated to the right side of the intestine, posterior gonad to the left side of the intestine. Tail conico-cylindrical, about 6.5 times the anal body diameter. The cylindrical portion of the tail represents about 24% of its total length.

Diagnosis. Molgolaimus sigmoides sp. nov. characterized by its body length (357–397 µm). Cuticle finely annulated. Head slightly set off. Four short cephalic setae (<two µm long) located at the level of buccal cavity and slightly anterior to head constriction. Amphidial fovea occupying 50–58% of the corresponding body diameter in the males and 39–44% in the female, located at about 1.5–2 times the head diameter. Buccal cavity with three small teeth, one dorsal and two ventrosublateral, the dorsal is slightly larger. Muscular pharynx with conspicuous spherical posterior bulb (80–85% of the corresponding body diameter). Pharyngeal lumen slightly cuticularized, except in the pharyngeal bulb where the valves are more cuticularized. Spicules S-shaped (2–2.2 times the cloacal body diameter). Gubernaculum surrounding the spicules at the distal end and with dorsal-caudal apophysis. Tail conico-cylindrical which corresponds to 4–6.5 times the cloacal or anal body diameter.

Differential diagnosis. Molgolaimus sigmoides sp. nov. resembles Molgolaimus sapiens Fonseca, Vanreusel & Decraemer, 2006 and Molgolaimus xuxunaraensis Fonseca, Vanreusel & Decraemer, 2006 mainly due to the peculiar shape of the spicules (S-shaped), precloacal supplements absence and by present short cephalic setae (two µm long in M. sapiens; <two µm long in M. sigmoides sp. nov. and not see in M. xuxunaraensis). However, M. sigmoides sp. nov. it is the only species of Molgolaimus that has a gubernaculum with a dorsal-caudal apophysis. This characteristic differentiates the new species both from those morphologically closest to it (M. sapiens and M. xuxunaraensis) and from the other valid species of the genus.

Type material. Five males and two females found. Holotype male (MOUFPE 0028), paratype female 1 (MOUFPE 0029), 4 male paratype (498–501 NM LMZOO-UFPE) and paratype female 2 (502–503 NM LMZOO-UFPE).

Type locality. South Atlantic Ocean, Continental shelf of the State of Pernambuco, Brazil, station 9 (S08°38′20.46″  W34°45′45.12″), November 26, 2019, 47 m.

Locality of other paratypes. South Atlantic Ocean, Continental shelf of the State of Alagoas, Brazil, station 11 (S09°15′30.54″  W34°57′13.14″), November 26, 2019, 87 m.

Etymology. The species name refers to similarity with species Molgolaimus longispiculum (Timm, 1961) Jensen, 1978.

Holotype male. Body cylindrical 811.5 µm long. Maximum body diameter corresponding about 4 times the head diameter. Cuticle faintly striated. Small papillae present throughout the body without a distribution pattern (observed in Paratypes 3 and 4, SEM photographs). Head off from the rest of the body by a constriction. Inner and outer labial sensilla indistinct. Four setiform cephalic setae (about 0.4 times the head diameter) located behind the head constriction (about six µm from anterior end). Amphidial fovea circular, located nine µm from anterior end (about 1.3 times the head diameter) and occupying 43% of corresponding body diameter. A small papilla associated with a cuticular pore present on both lateral edges of each amphidial fovea (distinctly visible in Paratypes 3 and 4, SEM photographs). Buccal cavity small, narrow, with slightly cuticularized walls. Three small teeth, a slightly larger dorsal tooth and two smaller ventrosublateral. Pharynx muscular (107.5 µm long), surrounding buccal cavity, consisting of narrow, cylindrical anterior portion and with conspicuous pyriform posterior bulb (78% of corresponding body diameter). Pharyngeal lumen slightly cuticularized, except in the pharyngeal bulb where the valves are more cuticularized. Nerve ring situated at 65% of the pharynx length from anterior end. Secretory-excretory pore not observed. Ventral gland located posterior of the pharyngo-intestinal junction. Cardia partially surrounded by intestine. Reproductive system with single anterior outstretched testis located ventrally to the intestine (germinal zone slightly to the right of the intestine). Spicules thin and elongated (6.9 times the cloacal body diameter). Gubernaculum funnel-shaped surrounding the spicules at the distal end and with anteriorly oriented apophysis. Precloacal supplements absent. Three caudal glands. Tail conico-cylindrical, about five times the cloacal body diameter. The cylindrical portion of the tail represents about one third of its total length. Spinneret short.

Paratype female. Similar to male. Body measuring 790.5 µm in length, with a maximum diameter of 36 µm (about five times the head diameter). Cephalic setae corresponding 0.5 times the head diameter. Amphidial fovea occupying 55% of corresponding body width and located nine µm from anterior end. Basal bulb occupying 80% of the corresponding body diameter. Nerve ring situated at 64% of the pharynx length, from anterior end. Secretory-excretory pore and ventral gland not observed. Vulva located 367.5 µm from anterior end, at 46% of body length. Reproductive system didelphic, with reflexed ovaries. Anterior gonad situated to the left side of intestine, posterior gonad to the right side of intestine. Tail conical with cylindrical terminal portion, about four times the anal body diameter. The cylindrical portion of the tail represents about 38% of its total length. Spinneret short.

Diagnosis. Molgolaimus paralongispiculum sp. nov. characterized by its body length (693–811.5 µm). Cuticle finely annulated. Head set off. Four setiform cephalic setae (about 0.4–0.5 times the head diameter) located behind the head constriction. Amphidial fovea occupying 55–60% of the corresponding body diameter, located at about 0.9–1.8 times the head diameter. Buccal cavity with three small teeth, one dorsal and two ventrosublateral, the dorsal is slightly larger. Muscular pharynx with conspicuous pyriform posterior bulb (76–83% of the corresponding body diameter). Pharyngeal lumen slightly cuticularized, except in the pharyngeal bulb where the valves are more cuticularized. Spicules thin and elongated (6.1–7.1 times the cloacal body diameter). Gubernaculum surrounding the spicules at the distal end and with anteriorly oriented apophysis. Tail conico-cylindrical (4–5 times the cloacal or anal body diameter).

Differential diagnosis. Molgolaimus paralongispiculum sp. nov. and four other species (Molgolaimus longispiculum; Molgolaimus tenuispiculum Ditlevisen, 1921; Molgolaimus gigasproximus Fonseca, Vanreusel & Decraemer, 2006 and Molgolaimus gigaslongincus Fonseca, Vanreusel & Decraemer, 2006) are the only representatives of the genus that have spicules >100 µm in length. Of these, M. longispiculum and M. paralongispiculum sp. nov. do not possess precloacal supplements, which helps to distinguish them from the other species mentioned above.

Considering only male specimens (female paratype described for M. longispiculum is immature), M. paralongispiculum sp. nov. shares the following features with M. longispiculum: de Man’s ratios b (7.4 in M. longispiculum and 6.5–8 in the new species), c (8.2 in M. longispiculum and 8–9 in M. paralongispiculum sp. nov.) and c’ (4.7 in M. longispiculum and 4–5 in M. paralongispiculum sp. nov.); total body length (737 µm long in M. longispiculum and 693–811.5 µm long in M. paralongispiculum sp. nov.) and the length of the cephalic setae (3 µm long in M. longispiculum and 3–3.5 µm long in M. paralongispiculum sp. nov.). However, the new species differs from M. longispiculum in relation to: position of the cephalic setae (cephalic setae positioned after the head constriction and below the level of the buccal cavity in M. paralongispiculum sp. nov. vs positioned close to the head constriction and at the same level of the buccal cavity in M. longispiculum); tail shape (conico-cylindrical in the new species vs conical with slightly swollen tip in M. longispiculum); de Man’s ratio a (22–27 in M. paralongispiculum sp. nov. vs 35.5 in M. longispiculum) and the shape of the gubernaculum (surrounding the spicules at the distal end and with anteriorly oriented apophysis in the new species vs lamellar in M. longispiculum).

Type material. One male and one female found. Holotype male (MOUFPE 0030) and paratype female (MOUFPE 0031).

Type locality. South Atlantic Ocean, Continental shelf of the State of Bahia, Brazil, station 23 (S13°04′10.32″ W38°25′46.98″), November 12, 2019, 65 m.

Locality of paratype female. South Atlantic Ocean, Continental shelf of the State of Pernambuco, Brazil, station 10 (S08°56′36.78″W34°50″16.02″), November 26, 2019, 54 m.

Etymology. The specific epithet of the species name is due to its relatively short spicules length. Latin brevis: short in length.

Holotype male. Body cylindrical 465 µm long. Maximum body diameter corresponding to about 4 times the head diameter. Cuticle faintly striated. Somatic setae not observed. Head slightly set off from the rest of the body by a slight constriction. Inner and outer labial sensilla indistinct. Four cephalic setae (about 1.3 times the head diameter) located behind the head constriction (three µm from anterior end). Amphidial fovea circular, located 10 µm from anterior end (about 2.4 times the head diameter) and occupying 55% of corresponding body diameter. Buccal cavity tubular, narrow, with slightly cuticularized walls. Teeth not observed. Pharynx muscular (70.5 µm long), surrounding buccal cavity, consisting of narrow, cylindrical anterior portion and with conspicuous spherical posterior bulb (81% of corresponding body diameter). Pharyngeal lumen slightly cuticularized. Nerve ring situated at 66% of the pharynx length from anterior end. Secretory-excretory system short. Secretory-excretory pore located just below the nerve ring (about 72% of the pharynx length). Ventral gland located posterior to the pharyngo-intestinal junction. Cardia not observed. Reproductive system with single anterior outstretched testis to the left of the intestine. Short spicule, with slightly cephalized anterior end (1 time the cloacal body diameter). Gubernaculum and precloacal supplements absent. Tail conical, about 5 times the cloacal body diameter.

Paratype female. Similar to male. Body measuring 633 µm in length, with a maximum diameter of 17 µm (3.4 times the head diameter). Amphidial fovea, occupying 50% of corresponding body width and located 12 µm from anterior end. Basal bulb occupying 84% of the corresponding body diameter. Nerve ring situated at 65% of the pharynx length, from anterior end. Secretory-excretory pore located just below the nerve ring (about 68% of the pharynx length). Ventral gland located posterior to the pharyngo-intestinal junction. Vulva located 298 µm from anterior end, at 47% of body length. Reproductive system didelphic, with reflexed ovaries. Anterior ovary situated to the right side of the intestine, posterior ovary to the left side of the intestine. Tail conical, about six times the anal body diameter.

Diagnosis. Molgolaimus brevispiculum sp. nov. characterized by its body length (465–633 µm). Cuticle finely annulated. Head slightly set off. Four cephalic setae (0.9–1.3 times the head diameter) located behind the head constriction. Amphidial fovea occupying 50–55% of the corresponding body diameter, located at about 2.4 times the head diameter to the anterior end. Buccal cavity unarmed. Muscular pharynx with cuticularized lumen and conspicuous spherical posterior bulb (81–84% of the corresponding body diameter). Secretory-excretory system short. Secretory-excretory pore located just below the nerve ring. Short spicule, with slightly cephalized anterior end (one time the cloacal body diameter). Gubernaculum and precloacal supplements absent. Tail conical (five to six times the cloacal or anal body diameter).

Differential diagnosis (Table 6). Only males of Molgolaimus typicus (Furstenberg & Vincx, 1992) and Molgolaimus drakus (Fonseca, Vanreusel & Decraemer, 2006) have been described, thus the comparison between those species and Molgolaimus brevispiculum sp. nov. is only based on males. The absence of gubernaculum is a common characteristic between M. typicus, M. drakus and M. brevispiculum sp. nov., which are thus different from the other species of the genus Molgolaimus. Nonetheless, other features as the absence of precloacal supplements, the spicules length, the cephalic setae length, the size of the amphidial fovea in relation to the body diameter and the de Man’s ratio help in differentiating those three species (Table 6). In addition, M. brevispiculum sp. nov. differs from M. typicus with regard to the position of the secretory-excretory pore, with this structure located at the same level as the amphidial fovea in M. typicus and just below the nerve ring in the new species.